The technology of recombinant DNA manipulation has evolved to a point which now permits the genetic engineering of some higher organisms. In the area of plant technology, it has become possible to genetically engineer many crop plant species, including several commercially important crops. Now that it is possible to transfer exogenous genetic traits into plants, a logical area of investigation is to identify traits which can be added to the plants which will increase their agricultural value. Since the biochemical mechanisms responsible for plant vigor and growth are poorly characterized and understood, and since the technology of the genetic transfer of exogenous traits into plants permits only a relatively few genes to be transferred into plants at any one time, it would appear difficult to envision, given the present state of the art, how dramatic changes in inherent plant growth or vigor characteristics can be obtained through genetic engineering. However, since actual crop yields in normal field situations are often dictated more by predatory factors on the plants, such as disease or insect predation, it is possible to increase the actual yield of crop plants under field conditions not by making the plants grow better, but by adversely effecting the limiting predatory factors which would otherwise decrease the effective yield from the plants. The present techniques for controlling insect predation of crop plants are based on use of synthetic chemical insecticides, which have been an accepted component of the cultivation practices for major agricultural crops, at least in the developed countries, for several decades. Most of the major agricultural chemicals utilized for insecticidal purposes are toxic to a relatively broad spectrum of insect pests and many persist in the environment giving rise to adverse environmental consequences. There have therefore been many efforts to develop pesticidal compounds that are uniquely toxic to specific insects and which also do not persist in the environment.
Biological insecticidal agents can meet several of these criteria For example, there have been several products based on the use of various forms of the delta-endotoxin produced by the soil dwelling microorganism Bacillus thuringiensis (B.t.) as insecticidal agents. This polypeptide toxin has been found to be specifically toxic to Lepidopteran insects, and has been used for many years commercially as a foliar applied insecticide. It has also recently been found that various forms of the B.t. toxin can be toxic to insects, when expressed inside the tissues of plants on which the insects feed. This is the first, and so far only, example of a natural biological insecticide being expressed in the cells of transgenic plants.
It has also been a feature of the history of the use of insecticide in agricultural applications that the insects sometimes become resistant to the applied pesticides. This phenomenon occurs through natural selection of the most insecticidal resistant members of the insect population following continual application of a single insecticidal agent year after year. While no broad spectrum resistance in insects has yet been shown to have developed to a biological toxin, such as the B.t. delta-endotoxin, the possibility of the development of such a resistant population must be considered in the long-term planning for the development of insect resistant plants. One way to minimize the possibility of the development of any such insecticidal resistant populations is to impose the insect predators to a regimen of at least two toxins, each of which has an independent mode of activity. The use of two agents, either simultaneously or sequentially imposed on the target populations, dramatically decreases the statistical possibility that resistant insect populations could develop. To be useful as a biological agent to be expressed in plants, such toxins should preferably be polypeptides that can be introduced into plant cells through single gene traits. One place to look for a source for such a toxin is animals which are insect predators.
One class of organism which is able to predate on insects is the scorpion. The scorpion is an Arthropod predator which has relatively poorly developed senses, but which has developed the ability to create a venom which rapidly immobilizes its insect prey, which is otherwise more mobile than the scorpion. The venom of the scorpion serves a dual function, as a defense against possible scorpion predators and to procure its own prey. The venom is therefore composed of a complex cocktail of toxins, many of which are pharmacology active proteins. The activity of the individual proteins from the scorpion toxin has been found to be relatively selective toward specific classes of organisms, such as mammals, insects, or crustaceans.
Several insect-selective neurotoxins have been isolated from scorpions and have been characterized and sequenced. One such neurotoxin, the peptide AaIT which has been isolated from the North African scorpion Androctonus australis Hector, is a highly charged polypeptide consisting of seventy amino acids. It has been reported, based on in vitro studies, that the . specificity of AaIT peptide is toward a synaptosomal membrane vesicle of insects, and that the protein shows no affinity toward other insect tissues, or for any mammalian, arachnid or crustacean tissues. The AaIT polypeptide binds specifically, reversibly and with high affinity to a single class of non-interactive binding sites on insect neural membranes.
One difficulty incumbent in the genetic engineering of plants to express toxic compounds is that the toxicity of such compounds to plants cells can adversely effect the ability to recover transformed plants. It appears, for example, that plant cells imbued with the trait to produce the full-length amino acid sequence of the B.t. delta endotoxin are negatively selected during present transformation techniques, perhaps due to toxicity of the full-length toxin on the plant cells themselves. It is therefore the case, at least at present, that the in vivo toxicity to plant cells of peptides toxic to insects is not predictable. It is also not possible to predict the toxicity of insect toxins which are normally injected into the insect when such toxins are administered to insects by ingestion.